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Glycoblology vol. 7 no. 8 pp.

1085-1088, 1997

The effects of calystegines isolated from edible fruits and vegetables on mammalian liver glycosidases

Naoki Asano, Atsushi Kato, Katsuhiko Matsui, Alison A.Watson1, Robert J.Nash1, Russell J.Molyneux2, Lucy Hackett3, Joanna Topping3 and Bryan Winchester3'4
Faculty of Pharmaceutical Sciences, Hokunku University, Kanazawa, JapanJ 'Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, UK, Western Regional Research Center, ARS, USDA, Albany, CA, USA, and 3Division of Biochemistry and Genetics, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK "To whom correspondence should be addressed

The polyhydroxylated nortropane alkaloids called calystegines occur in many plants of the Convolvulaceae, Solanaceae, and Moraceae families. Certain of these alkaloids exhibit potent inhibitory activities against glycosidases and the recently demonstrated occurrence of calystegines in the leaves, skins, and sprouts of potatoes (Solatium tuberosum), and in the leaves of the eggplant (S.melongena), has raised concerns regarding the safety of these vegetables in the human diet. We have surveyed the occurrence of calystegines in edible fruits and vegetables of the families Convolvulaceae, Solanaceae, and Moraceae by GC-MS. Calystegines A3, Bl, B2, and Cl were detected in all the edible fruits and vegetables tested; sweet and chili peppers, potatoes, eggplants, tomatoes, Physalis fruits, sweet potatoes, and mulberries. Calystegines Bl and Cl were potent competitive inhibitors of the bovine, human, and rat B-glucosidase activities, with K, values of 150, 10, and 1.9 uM, respectively for Bl and 15,1.5, and 1 uM, respectively, for Cl. Calystegine B2 was a strong competitive inhibitor of the a-galactosidase activity in all the livers. Human B-xylosidase was inhibited by all four nortropanes, with calystegine Cl having a K, of 0.13 uM. Calystegines A3 and B2 selectively inhibited the rat liver B-glucosidase activity. The potent inhibition of mammalian B-glucosidase and a-galactosidase activities in vitro raises the possibility of toxicity in humans consuming large amounts of plants that contain these compounds. Key words: edible plants/calystegines/glycosidase inhibitors/ bovine, human, and rat liver

sess a configuration analogous to the C4, C3 and C2 OH groups and ring heteroatom of the a-glucosidase inhibitor 1deoxynojirimycin (DNJ) (Figure 1). These structural similarities suggested that calystegines might also have glycosidase inhibitory activities. Asano et al. (1994a,b, 1995) and Molyneux et al. (1993) have reported that calystegine B2 is a potent competitive inhibitor of plant p-glucosidases and a-galactosidases, and calystegines Bl and Cx of plant P-glucosidases but not of a-galactosidase. A particularly interesting aspect of the biological activity of calystegines is their potential toxicity toward animals or humans which may ingest them. As with other glycosidase inhibitors, phenotypes of specific lysosomal storage diseases may be produced, each depending upon the identity of the glycosidase which is inhibited (Dorling, 1984). In the case of swainsonine from Astragalus, Oxytropis, and Swainsona species (Molyneux and James, 1982) the signs of poisoning are analogous to those of genetic a-mannosidosis, a rare disease in humans but not uncommon in Angus cattle (Dorling et al, 1978). Castanospermine (Hohenschutz et ai, 1981), which occurs in seeds of the Moreton Bay chestnut (Castanospermum australe), is a potent inhibitor of a-glucosidase, and signs of poisoning of livestock and humans are similar to those observed for Pompe disease, which arises from a genetic deficiency of the same enzyme (Saul et al., 1985; Molyneux et al., 1994). Therefore, it is possible that the calystegines, which inhibit (3-glucosidase and a-galactosidase, might produce syndromes that mimic a genetic deficiency of such activities, namely, Gaucher and Fabry disease, respectively (Dorling, 1984; Molyneux et al, 1994). The demonstrated occurrence of calystegines in the leaves, skins, and sprouts of potatoes (Solarium tuberosum), and in the leaves and fruits of the egg plant (S.melongena) (Nash et al., 1993; Drager et al, 1995), raises concerns regarding the safety of these vegetables in the human diet In this article we report a survey of the occurrence of calystegines in edible fruits and vegetables and the inhibitory activities of calystegines against bovine, human, and rat liver glycosidases. Results The occurrence of calystegines in edible fruits and vegetables

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Introduction The tropane alkaloids are bicyclic amines which combine pyrrolidine (five membered) and piperidine (six-membered) rings in a bridged structure. Recently, polyhydroxylated nortropane alkaloids, named calystegines, have been isolated from plants in the Convolvulaceae, Solanaceae and Moraceae (Tepfer et al., 1988; Goldman et al., 1990; Nash et al, 1993; Asano et al, 1994a,b). The C2, C3, and C4 OH groups and ring heteroatom in the six-membered ring of (+)-calystegine B2 (Figure 1) pos Oxford University Press

The edible fruits and vegetables in the families Solanaceae, Convolvulaceae, and Moraceae were analyzed for calystegines by GC-MS, and the results are summarized in Table I. The tetrahydroxylated calystegine B2 was present in varying amounts in all of the species analyzed, but particularly high concentrations were found in commercially available sweet peppers, eggplants, and sweet potatoes in Japan. In those species which were found to contain more than one calystegine, the concentration of calystegine B2 was generally higher than those of the other polyhydroxylated nortropanes. Only 6 of the 1085

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HN

Calystegine A3

Calystegine

HN

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Calystegine B2

Calystegine C,

HN HOH2C
1-Deoxynojirimycin
Fig. 1. Effects of calystegines on liver glycosidases.

11 species analyzed contained the trihydroxylated calystegine A3, the highest concentrations being found in tomatoes and potatoes. Similarly, five species contained calystegine Bl. However, the distribution of the pentahydroxylated calystegine Cl was more limited as this compound was only found in sweet peppers, cape gooseberries, and sweet potatoes. The variability of both the type and concentration of calystegines detected in potatoes, eggplant, and sweet potatoes analyzed in Japan and the United Kingdom could be due to varietal or climatic differences (Table I). Inhibitory effects of calystegines toward mammalian liver glycosidases The effects of the calystegines A3, Bl, B2, and Cl, found in edible fruits and vegetables, on mammalian liver glycosidases were investigated by assaying 12 activities using synthetic substrates. The ICJO and Kj values for the calystegines that inhibited glycosidases are shown in Table n. Calystegines Bl and Cl competitively inhibited the (3-glucosidase activity in all three species, whereas A3 and B2 only inhibited the rat liver (3-glucosidase, suggesting that the calystegines mimic the active site of the rat enzyme to a greater degree than those of the bovine and human enzymes. Purified human lysosomal fi-glucosidase was completely inhibited by calystegine B1 at 1 \LM concentration. Calystegines Bl and Cl have been shown pre1086

viously to be potent competitive inhibitors of almond P-glucosidase with Kj values of 1.8 and 0.45 \iM, respectively (Asano etal, 1995). In contrast, human (3-xylosidase was more vulnerable to inhibition than the rat liver enzyme and was inhibited by all four compounds. Calystegine B2 inhibited ot-galactosidase from all 3 species with the rat enzyme again being the most severely affected. Calystegine A3 was a moderate inhibitor of human ot-galactosidase but did not inhibit the bovine or rat enzymes. The a-galactosidase activity in all these species was unaffected by calystegines Bl and Cl. The P-galactosidase activity in the crude human liver-extract was inhibited by 85% by lmM calystegine Bl. When the human lysosomal {J-galactosidase was separated from the broad specificity, cytosolic P-glucosidase / P-galactosidase, it was only weakly inhibited (40% at lmM), indicating that the cytosolic enzyme contributes to the total P-galactosidase activity in the human liver extract and is inhibited by Bl. Calystegine Bl inhibits bovine liver p-galactosidase competitively (Kj, 1.6 |xM) (Asano et al, 1995).

Discussion The concentrations of calystegines in the edible fruits and vegetables tested in this work are relatively low. However, Drager et al. (Drager et al., 1995) have reported that the calystegine

Effects of calystegines on liver glycosidases

Table L Calystegine content of edible fruits and vegetables Plant Means of acquisition' Detected calystegines B, u.g/g fresh weight Solanaceae Capsicum annum var. angulosum (sweet peppers) Capsicum fnaescens (chili peppers) Cyphomandra betaceae (tomatillo) Lycopersicum esculentum (tomatoes) Physalis peruviana (cape gooseberries) Pkysalis exocarpa (tamarillo) Solanum melongena (egg-plants) Solanum scabrum (huckleberries) Solanum tuberosum (potatoes) Convolvulaceae Ipomoea batatas (sweet potatoes) Moraceae Morus alba (mulberries) A B C B C C A C B A B A C None 0.235 None 1.1 0.003 None None 0.312 None None 1.17 None 0.11 None 12 Trace None None 0.038 None 7 None None None None 16 2.37 None 37 0.269 0.002 4.5 0.048 0.002 73 0.473 0.007 7 2.22 19 1.12 4 None None None 0.005 None None None None None None 9 0.61 Downloaded from glycob.oxfordjournals.org by guest on October 27, 2010 None

c,

'A, Shop-bought in Japan. B, Grown at Institute of Grassland and Environmental Research, UK. C, Shop-bought in UK. Vg/g dry weight

content varies greatly within one plant and that the calystegine B2 content is 400 jig/g tissue in sprouts growing from greening potato tubers that were kept in daylight for 2 weeks, compared with 7 u,g/g for the whole plant. Nash et aL (Nash et al, 1993) found calystegine levels of 0.01% of the skin fresh weight of the potato cultivar "Estima," while the concentration of the rest of the tuber was one-tenth of this amount. It is noteworthy that there have been periodic reports of gastrointestinal disturbances and occasional deaths associated with consumption of greened potatoes and potato skins, and that potato leaves and sprouts are eaten in Pakistan. We have found that the calystegine contents in the edible parts of egg plants in the United Kingdom were very low, whereas the levels of calystegines A3, Bl, and B2 in the leaves were 14, 34, and 194 u,g/g dry leaves, respectively. The calystegine content appears to vary greatly among cultivars because the shop-bought eggplants in Japan contained a calystegine B2 level of 73 n-g/g fresh weight. The introduction of a hydroxyl group at Cdexo in calystegines A3 and B2 to give Bl and Cl, respectively, markedly

enhances the inhibition of the lysosomal (3-glucosidase in all three species and of the human and rat (3-xylosidase. This suggests that the hydroxylated pyrrolidine ring is the key structural determinant for inhibition of p-glucosidase and (3-xylosidase. In contrast, the addition of this group abolishes the inhibition of bovine, human, and rat a-galactosidase by calystegine B2 and of human a-galactosidase by calystegine A3. It is difficult to understand the structural basis for the selective inhibition of a-galactosidase by calystegine B2 but the configuration at carbons 1, 4, and 5 is the same as in carbons 1, 4, and 5 of a-galactose although the substituents at C2 and C3 are in the wrong configuration. The configuration of the hydroxyl at C6 must be inappropriate. It would be interesting to test the C6 epimer of calystegine Cl for inhibition of a-galactosidase. Potent glycosidase inhibitors do not need to be present at high concentrations for toxic effects. In the case of the a-mannosidase inhibitor, swainsonine, the low concentration ingested is made more effective by its ability to permeate the plasma and lysosomal membranes freely, and to become concentrated

Table IL Effects of calystegines on mammalian liver lysosomal glycosidases Compound K V and (KI, M-M) P-Glucosidase Bovine Calystegine A 3 Calystegine B, Calystegine B 2 Calystegine C, NT" 360 (Ki, 150) NI 60 (Ki, 15) Human Nl 50 (Ki, 10) Nl 3 (Ki, 1.5) Rat 90 (Ki, 24) 4.6 (Ki, 1.9) 15 (Ki, 8.9) 3.6 (Ki, 1) a-Galactosidase Bovine Nl Nl 270 (Ki, 72) Nl Human 410 Nl 140 (Ki, 185) Nl Rat NI Nl 21 (Ki, 4.8) Nl P-Xylosidase Human 400 50 (Ki, 19) 100 (Ki, 60) 5 (Ki, 0.13) Rat 850 120 Nl NI

Concentration giving 5 0 % inhibition. ""Less than 5 0 % at 1 mM.

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inside the lysosomes because it becomes protonated due to the low pH (Chotai et al, 1983). A threshold of toxicity is difficult to establish but a conservative estimate of the concentration of swainsonine in the diet which should be of concern could be as low as 0.001% of the dry weight of the plant (Molyneux et al., 1994). As shown in this work, the presence of calystegines in human foods such as tomatoes, potatoes, eggplants, and sweet potatoes poses the question as to the effect that these compounds might have on humans. Furthermore, it has been established in this work that there are differences between mammalian groups in the susceptibility of their liver glycosidases to inhibition by calystegines. Although, calystegines Bl and Cl potently inhibited human liver lysosomal fi-glucosidase, preliminary experiments have indicated that they do not cause additional lysosomal storage in human fibroblasts in culture for one week in the presence of 1 mM of the calystegine (B. Winchester, unpublished observations). Animal feeding experiments with the pure calystegines are being carried out. Materials and methods
Extraction of calystegines from plant materials for identification Fresh plant materials (in the case of mulberries, dried fruits) were homogenized with 50% MeOH or EtOH in a Waring blender and allowed to stand for 24 h at room temperature. The mixture was filtered and centrifuged. The supernatant was applied to a column of Amberlite IR-120B (H+ form) and the adsorbed fraction, after elution with 0.5 M NH 4 0H, was further applied to a column of Dowex 1-X2 (OH'foim) and eluted with water. The water eluate was concentrated and lyophilized. GC-MS analysis Samples were dried and silylated using 100 (xl of Sigma SIL-A (Sigma Chemical Co.). The samples were heated at 30C for 30 min and then 0.3 u.1 was injected directly onto a GC column (25 m x 0.25 mm BPX5 capillary column, SGE). The temperature program ran for 25 min, 18O-3OOC with an initial rate of increase of 10C per min and was then held at 300C. The mass spectrometer was a QMASS 910 (Perkin-Elmer) with the El mass range set at 100-650 amu. The identity and quantification of the calystegines were assessed by GC-MS of reference samples. Enzyme assays The partially purified lysosomal fraction prepared by the procedures of (Tsuji et aL. 1977) was used as a source of rat or bovine liver lysosomal glycosidases. The lysosomal glycosidase activities were assayed using the appropriate pnitrophenyl glycosides (Sigma Chemical Co.) as substrates at a concentration of 2 mM in 0.05 M sodium acetate buffer, pH 5.0. The human liver extract was prepared according to the method of Winchester et aL (Winchester et aL, 1990). The glycosidase activities in the extract of human liver were assayed by using the appropriate fluorigenic 4-methylumbelliferyl glycoside substrate (Melford Laboratories) at a concentration of 0.5 mM, at the optimal pH for each enzyme (Burditt et aL, 1980). Enzyme inhibition modes and K, values of the calystegines were determined from the slope of Lineweaver-Burk plots, or by the Dixon procedure.

Containing sugars from Moms alba and their glycosidase inhibitory activities. Carbohydr. Res., 259, 243-255. Asano,N., Kato,A., Oseki.K., Kizu.H. and Matsui.K. (1995) Calystegins of Physalis alkekengi var.francheti (Solanaccae)structure determination and their glycosidase inhibitory activities. Eur. J. Biochem., 229, 369-376. Burditt.LJ., Chotai.K., Hirani.S., Nugent,P.G. and Winchester.B. (1980) Biochemical studies on a case of feline mannosidosis. Biochem. J., 189, 467473. Chotai.K., Jenrungs.C, Winchester.B. and Dorling,P. (1983) The uptake of swainsonine, a specific inhibitor of ot-D-mannosidase, into normal human fibroblasts in culture. J. Cell Biochem., 21, 107-117. Dorling,P.R. (1984) Lysosomal storage diseases in animals. In DingleJ.T., Dean,R.T. and Sly.W. (eds.), Lysosomes in Biology. Elsevier, Amsterdam, pp. 347-379. Dorling^.R-. Huxtable.C.R. and Vogel.P. (1978) Lysosomal storage in Swamsona spp. Toxicosis: an induced mannosidosis. Neuropath. AppL NeurobioL, 4, 285-295. Drager.B., van Almsick^A. and Mrachtz,G. (1995) Distribution of calystegines in several Solanaceae. Planta Med., 61, 577-579. Goldmann.A., MilatJvl.L., Ducrot.P.H., LallemandJ.Y., Maille.M., Lepingle,A., Charpin.I. and Tepfer.D. (1990) Tropane derivatives from Calystegin sepium. Phytochemistry, 29, 2125-2127. Hohenschutz,L.D., Bell^E.A., Jcwess,P.J., Leworthy.D.P., Pryce,RJ., Amold,E. and ClardyJ. (1981) Castanospermine. Phytochemistry, 20, 811-814. Molyneux.RJ., and James,L.F. (1982) Loco intoxication: indolizidine alkaloids of spotted locoweed (Astragalus lentiginosus). Science, 216, 190-191. Molyncux.RJ., Pan.Y.T., GoldmannA, Tepfer.D.A. and ElbeirvA.D. (1993) Calystegins, a novel class of alkaloid glycosidase inhibitors. Arch. Biochem. Biophys., 304, 81-88. Molyneux.R.J., James.L.F., Ralphs.M.H., Pfister.J.A., Panter.K.E. and Nash.RJ. (1994) Polyhydroxylated glycosidase inhibitors from poisonous plants of global distribution: analysis and identification. In Colegate.S.M. and Dorling.P.R. (eds.), Plant-Associated ToxinsAgricultural, Phytochemical and Ecological Aspects. CAB International, Wallingford, U.K., pp. 107-112. Nash,RJ., Rothschildjd., Porter.E.A., Watson,A.A., Waigb,R.D. and Waterman .P.G. (1993) Calystegines in Solanum and Datura species and the death's-head hawk-moth (Acherontia Atropus). Phytochemistry, 34, 1281 1283. Saul.R., GhidoniJJ., Molyneux,RJ. and FJbein^A.D. (1985) Castanospermine inhibits a-glucosidase activities and alters glycogen distribution in animals. Proc. Natl. Acad. ScL, USA, 82, 93-97. TepferJD.A., Goldmann,A., Pamboukdjian.N., Maillejvi., Lepingle,A., Chevalier J)., Denaire, J. and Rosenberg, C. (1988) A plasmid of Rhizobium meliloti 41 encodes catabolism of two compounds from root exudate of Calystegium sepium. J. Bacterial., 170, 1153-1161. Tsuji,H., Hattori,N., Yamamoto.T. and Kato.K. (1977) The synthesis of rat liver lysosomes. 1. Comparison of the microsomal, Golgi and lysosomal P-glucuronidases J. Biochem. (Tokyo), 82, 619-636. Winchester.B., Barker.C, Baines.S., Jacob.G.S., Namgoong.S.K. and Fleet,G. (1990) Inhibition of a-L-fucosidase by derivatives of deoxyfuconojirimycin and deoxymannojirimycin. Biochem. J., 265, 277-282. Received on January 7, 1997; revised on March 24, 1997; accepted on April 17. 1997

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Acknowledgments
The work in the United Kingdom was partly funded by the Ministry of Agriculture, Fisheries and Food and the Wellcome Trust. The work in Japan was partly supported by the Special Research Fund of Hokuriku University. We thank Professor Brian Lake for carrying out examination by electron microscopy of cultures of human fibroblasts grown in the presence of calystegines.

References
Asano,N., Tomioka,E., Kizu.H. and Matsui.K. (1994a) Sugars with nitrogen in the ring isolated from the leaves of Morus bombycis. Carbohydr. Res., 253, 235-245. Asano.N., Oseki.K., Tomioka.E., Kizu.H. and Matsui.K. (1994b) N-

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